Gene Therapy for Diabetic Dogs
Overview Diabetes Mellitus is one of the leading cause of mortality and comorbidity in America, and although the disease can be prevented and managed there is no finite cure. Consistent Insulin therapy allows patients to stay on top of the disease by maintaing blood glucose levels. Insulin treatments, however, fail to prevent the complications that can arise from the condition. Insulin injections are also not suitable for all patient sbecause of the potential for hypoglycemia due to excess dosage. Currently there are many novel treatments being studied, but the one shown to be effective in dogs involves the genetic modification of skeletal muscle cells so that they may act as both a glucose sensor and a producer of insulin. In this study 6 12-month old male Beagles were given experimental diabetes by intravenous injections of a mixture of streptozotocin and alloxan which are cytotoxic to beta islet cells. Insulin production was now inhibited and hyperglycemia developed in all of the dogs. Glucokinase activity, oral glucose tolerance test, hypoglycemia during excericsie, and hormone and metabolite concentrations ins serum were used as tests to measure the progression of the disease and after treatment the reversal of the disease state. These dogs represent the first large animals to experience long-term correction of diabetes using gene transfer. Gene Expression The gene therapy used in this study is based on introducing a new gene to skeletal muscle cells. The delivery of the transgenes to the dogs was done via exposure of the host skeletal muscle cells to viral vectors on which the genes were expressed. An engineered human insulin gene (Ins) and rat glucokinase gene (Gck) were encoded onto an adeno-associated viral vector of serotype I (AAVI) via triple transfection of HEK293 cells. AAVIs are useful in gene therapy because of their wide range of tissue specificity, relative safety, and ability to infect and be expressed in non-dividing cells. Infection of human cells with an AAVI does not initiate viral replication, but rather the viral DNA is uncoated, converted into the duplex form, and integrated into a specific point on the human genome. The dogs underwent one-time intramuscular injection of the AAVI to 12-25 sites on the lateral thigh and the craneolateral face of the leg on both hind limbs, resulting in the expression of Gck and Ins by skeletal muscle cells. Skeletal muscle cells were chosen because they are the site for the uptake for approximately 70% of circulating glucose after a meal. The expression of Gck facilitates uptake of glucose when blood glucose is high. The expression of Ins by cells initiates the production of insulin from glucose absorbed from the blood. The presence of insulin enhances glucose absorption as well because glucose transporter type 4 (GLUT4) is controlled by insulin stimulation. This is vital for the idea of a glucose sensor because the insulin being produced to regulate GLUT4 is only being made when blood glucose is high and GLUT4 is active regulating glycemia. Response to Treatment The patients experienced normalization of fasting glycemia, accelerate disposal and uptake of glucose after diagnosis with an oral food challenge, and no episodes of hypoglycemia during excercise for greater than four years after expression of the transfer gene. These physiological correction were subsequently associated with appropriate decrease in body weight, reduced glycosylated plasma protein levels, and increased survival absent of secondary complications. It is also important to note that expression of either Gck or Ins alone does not result in correction of symptoms suggesting that co-expression is necessary for effective treatment. The synergistic expression of these two genes presents a form of glycemia regulation that is more effective and permanent than insulin therapy. Drawbacks to Treatment The main limitation of this study is that the experimentally induced diabetes in the dogs does not match immunological state of humans who what type 1 DM. Type 1 DM is also caused by numerous different mutations, and in humans, before clinical trials are done, we are unsure if these mutations will inhibit insulin production in all cells or just beta islet cells. However, skeletal muscle cells are a location less likely to be affected by autoantibodies. At this point, transgenic skeletal muscle cells are poised to provide a safer and more effective treatment than other novel models such as stem cell proliferate beta islet cells or traditional insulin therapy. References 1.Treatment of Diabetes and Long-term Survival Following Insulin and Glucokinase Gene Therapy 2. Adeno-associate Viral Vectors for Gene Therapy☁